Whittaker P A, Wood L, Mathrubutham M, Anand R
University Clinical Biochemistry, Southampton General Hospital, United Kingdom.
Genomics. 1993 Feb;15(2):453-6. doi: 10.1006/geno.1993.1089.
A phage contig of 400 kb that extends from the brain-specific promoter at the 5'-end of the human dystrophin gene, through the muscle-specific promoter over 100 kb further downstream, and across most of intron 1 has been assembled. To achieve this, a yeast artificial chromosome (YAC) subcloning approach was used. Total DNA from a yeast strain containing a 400-kb YAC from the dystrophin gene was cloned using a lambda phage vector containing RNA polymerase promoters flanking the cloning sites. Phage containing human DNA inserts were then ordered into an overlapping set by hybridization of end-specific RNA probes from individual clones back to plaque lifts of gridded phage subclones. The clones generated will be useful as reagents for detailed structural and functional analyses of this region of the dystrophin gene.
一个400 kb的噬菌体重叠群已经组装完成，它从人类肌营养不良蛋白基因5'端的脑特异性启动子开始，延伸至下游100 kb以外的肌肉特异性启动子，并跨越了大部分内含子1。为实现这一目标，采用了酵母人工染色体（YAC）亚克隆方法。使用一个在克隆位点两侧带有RNA聚合酶启动子的λ噬菌体载体，对来自含有肌营养不良蛋白基因400 kb YAC的酵母菌株的总DNA进行克隆。然后，通过将各个克隆的末端特异性RNA探针与网格化噬菌体亚克隆的菌斑杂交，将含有人类DNA插入片段的噬菌体排列成一个重叠组。所产生的克隆将作为试剂，用于对肌营养不良蛋白基因这一区域进行详细的结构和功能分析。
